The broad, long-term goal of the research conducted under DE-10510 is to understand, at the molecular level, bacterial responses triggered by bacteria-host interaction; knowledge that is central to our comprehension of microbial pathogenesis and host defense. The first step in Porphyromonas gingivalis infection is colonization of the junctional epithelium lining the gingival sulcus. The objective of the proposal was to identify P. gingivalis genes and proteins that determined this initial interaction. A direct result of the study was the demonstration that gingipains were critical players in adherence of the bacterium to human epithelial cells. These important discoveries set the stage for new hypotheses that address the functional genomics of adhesion. Specific Aim 1 of this application tests the hypothesis that specific gingipain domains determine P. gingivalis adhesion to, and detachment from, epithelial cells. This will be tested with purified recombinant gingipain proteins and peptides in adhesion competition and blocking experiments. Specific Aim 2 addresses the hypothesis that a complex of proteins is involved in adhesion, and components of the complex will be identified. Specific Aims 3 and 4 introduce a new research area centered on the hypotheses that during infection a defined set of P. gingivalis genes are expressed to promote disease and survival of the organism in the face of host defenses; and the expression of non-essential genes is down-regulated to ensure the efficient use of cell resources. Thus, in Specific Aim 3, subtractive hybridization techniques will be used to examine gene expression after a defined stimulus, contact with epithelial cells. Specific Aim 4 continues this investigation with genome-wide analyses using state of the art P. gingivalis microarrays. Within the context of increased bacterial resistance to antibiotics, and the emergence of new human pathogens, an understanding of the molecular interactions between an infecting bacterium and its host is the accelerant to new drug discovery. Validation of our hypotheses regarding gingipains and epithelial cell adhesion may further stimulate searches for second-generation antibiotics directed to these proteinases. The discovery of new activities related to the pathogenic process will increase the repertoire of future targets, and drive the hunt for novel therapeutic interventions.